Name: assessment of mouse judgment bias through an olfactory digging task
Uploaded: 2022-03-04T14:00:00
Description: Watch this Scientific Journal Video about Assessment of Mouse Judgment Bias through an Olfactory Digging Task at JoVE.com

The authors are grateful to Miguel Ayala, Lindsey Kitchenham, Dr. Michelle Edwards, Sylvia Lam and Stephanie Dejardin for their contributions to the Reseasco et al.19 validation work which this protocol is based on. We would also like to thank the mice and our wonderful animal care technicians, Michaela Randall and Michelle Cieplak.

Experiments were approved by the University of Guelph&#39;s Animal Care Committee (AUP #3700), conducted in compliance with Canadian Council on Animal Care guidelines, and reported in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments)20 requirements.
1. Experiment preparation
<ol>
	<li>Experimental design (see Table 1). 
	NOTE: This behavioral test is a scent-based Go/Go digging task, in which mic...

<ol>
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The scent-based digging protocol and results outlined here demonstrate the ability of this novel JB task to detect changes in mouse affective state. The task thus presents a valuable tool for diverse fields of research. Similar to any JB task, to assess animal affect it is critical that animals first learn to discriminate between cues (step 4.7.3) and that the ambiguous stimulus is interpreted as intermediate (step 5.3). Though simple, meeting these requirements can be challenging, particularly in laboratory mice for whi...

Measuring affectively modulated judgment bias (henceforth JB) has proven to be a useful tool for studying the emotional states of animals. This innovative approach borrows from human psychology since humans experiencing positive or negative affective states (emotions and longer-term moods) reliably demonstrate differences in the way they process information1,2,3. For example, humans experiencing anxiety or depression might interpret neutral facial expressions as negative, or neutral sentences as threatening4,5. It is likely that these biases have an adaptive value and are therefore conserved across species6,7. Researchers aiming to assess animal affect have cleverly taken advantage of this phenomenon, operationalizing optimism as the increased expectation of reward in response to neutral or ambiguous cues, and pessimism as the increased expectation of punishment or reward absence8,9. Thus, in an experimental setting, optimistic and pessimistic responses to ambiguous stimuli can be interpreted as indicators of positive and negative affect, respectively10,11.
Compared to other indicators of animal affect, JB tasks have the potential to be particularly valuable tools since they are capable of detecting both the valence and intensity of affective states10,11. The ability of JB tasks to detect positive states (e.g., Rygula et al.12) is especially useful since most indicators of animal affect are limited to the detection of negative states13. During JB tasks, animals are typically trained to respond to a positive discriminative cue predicting reward (e.g., high-frequency tone) and a negative discriminative cue predicting punishment (e.g.,&#160;low-frequency tone), before being presented with an ambiguous cue (e.g., intermediate tone)8. If in response to ambiguous cues an animal &#39;optimistically&#39; performs the trained response for the positive cue (as if expecting reward), this indicates a positive judgment bias. Alternatively, if animals demonstrate the negative trained response to avoid punishment, this is indicative of &#39;pessimism&#39; or negative judgment bias.
Since the development of the first successful JB task for animals by Harding and colleagues8, several JB tasks have been developed for a wide range of species across diverse research fields7. But despite their increasing popularity, animal JB tasks are often labor-intensive. Moreover, perhaps because they are methodologically different from the human tasks that inspired them, they sometimes produce null or counterintuitive results14 and commonly yield only small treatment effect sizes15. As a result, JB tasks can be challenging to develop and implement. In fact, for laboratory mice, the most widely used vertebrates in research16,17 and a species heavily relied upon to model affective disorders18, only one JB task has been successfully validated as sensitive to changes in affective state19 despite many attempts over the past decade (see supplementary material of Resasco et al.19&#160;for a summary). This article describes the recently validated murine JB task, detailing its biologically relevant design, and highlighting the ways that this humane task can be applied to test important hypotheses relevant to mouse affect. Overall, the protocol can be implemented to investigate the affective effects of any variable of interest on JB in mice. This would include categorical treatment variables as described here (drug or disease effects, environmental conditions, genetic background, etc.), or relationships with continuous variables (physiological changes, home cage behaviors, etc.).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absolute ethanol</td>
			<td>Commercial alcohol</td>
			<td>P016EAAN</td>
			<td>Dilute to 70% with distilled water, for cleaning</td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Fischer</td>
			<td>55395</td>
			<td>15 mL tubes used to dilute essences here. However, size may be selected based on total volume required for sample size</td>
		</tr>
		<tr>
			<td>Cheerios (original)</td>
			<td>Cheerios</td>
			<td>N/A</td>
			<td>Commercially available. Used as reward to train animals to enter annex cage for handling</td>
		</tr>
		<tr>
			<td>Corncob bedding</td>
			<td>Envigo</td>
			<td>7092</td>
			<td>Teklad 1/8 corncob bedding used in digging pots and animal cages</td>
		</tr>
		<tr>
			<td>Cotton pads</td>
			<td>Equate</td>
			<td>N/A</td>
			<td>Commercially available. Modified in lab to fit within tissue cassettes for scent dispensing</td>
		</tr>
		<tr>
			<td>Digging pots</td>
			<td>Rubbermaid</td>
			<td>N/A</td>
			<td>Commercially available. Containers were modified to fit the apparatus in the lab</td>
		</tr>
		<tr>
			<td>Dried, sweetened banana chips</td>
			<td>Stock and Barrel</td>
			<td>N/A</td>
			<td>Commercially available. High value reward in JB task</td>
		</tr>
		<tr>
			<td>JB apparatus</td>
			<td></td>
			<td>N/A</td>
			<td>The apparatus was made in the lab</td>
		</tr>
		<tr>
			<td>JWatcher event recording software</td>
			<td>Animal Behavior Laboratory, Macquarie University</td>
			<td>Version 1.0</td>
			<td>Freely available for download online. Used to score digging behavior during JB task</td>
		</tr>
		<tr>
			<td>Mint extract</td>
			<td>Fleibor</td>
			<td>N/A</td>
			<td>Commercially &#34;Menta (Soluci&#243;n)&#34;. Discriminative stimulus odor</td>
		</tr>
		<tr>
			<td>Rodent Diet</td>
			<td>Envigo</td>
			<td>2914</td>
			<td>Teklad global 14% protein rodent maintenance diets. Low value reward in JB task and regular diet in home cage</td>
		</tr>
		<tr>
			<td>SAS statistical software</td>
			<td>SAS</td>
			<td>Version 9.4</td>
			<td>Other comparable software programs (e.g. R) are also appropriate</td>
		</tr>
		<tr>
			<td>Vanilla extract</td>
			<td>Fleibor</td>
			<td></td>
			<td>Commercially available &#34;Vainilla (Soluci&#243;n)&#34;. Discriminative stimulus odor</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Sony</td>
			<td>DCR-SX22</td>
			<td>Sony handycam.</td>
		</tr>
	</tbody>
</table>

assessment of mouse judgment bias through an olfactory digging task

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This phenomenon has long been observed in humans, with individuals in positive states responding to ambiguity 'optimistically' and those in negative states instead showing 'pessimism'. Researchers aiming to assess animal affect have taken advantage of these differential responses, developing tasks to assess judgment bias as an indicator of affective state. These tasks are becoming increasingly popular across diverse species and fields of research. However, for laboratory mice, the most widely used vertebrates in research and a species heavily relied upon to model affective disorders, only one JB task has been successfully validated as sensitive to changes in affective state. Here, we provide a detailed description of this novel murine JB task, and evidence of its sensitivity to mouse affect. Though refinements are still necessary, assessment of mouse JB opens the door for answering both practical questions regarding mouse welfare, and fundamental questions about the impact of affective state in translational research.

Results presented here reflect relevant findings from Experiment 1 of Resasco et al.19. Subjects in this experiment were 18 female C57BL/6NCrl (&#39;C57&#39;) and 18 Balb/cAnNCrl (&#39;Balb&#39;) mice. Animals arrived at the facility at 3-4 weeks of age and were randomly assigned to environmentally enriched or conventional housing treatments (EH or CH, respectively) in mixed strain quartets25. Each cage contained one C57 and one Balb, in addition to two DBA/2NCrl mice being...

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Assessment of Mouse Judgment Bias through an Olfactory Digging Task

This article provides a detailed description of a novel mouse judgment bias protocol. Evidence of this olfactory digging task's sensitivity to affective state is also demonstrated and its utility across diverse research fields is discussed.

Judgment biases (JB) are differences in the way that individuals in positive and negative affective/emotional states interpret ambiguous information. This ...

This is the first ever mouse judgment bias task to be validated as sensitive to changes in affective state, in other words, mood or emotion. It can be used to investigate the effects of any variable on mouse affect. This protocol uses an ethological design, making it easy for mice to learn, and it's also welfare friendly, since the task avoids the use of punishment or negative reinforcement.This task can assess the impact of husbandry or experimental procedures on mouse affective states, making it valuable across diverse fields, like animal welfare research or modeling human affective disorders. Assuming researchers are already comfortable with non-aversive handling methods, our main advice is to use pilots before training to identify appropriate odor mixtures, in other words, the ones that mice treat as ambiguous. This behavioral test is a scent-based go-go digging task in which a mouse must dig for high-or low-value food rewards.The experimental apparatus comprises a rectangular arena with two arms. One arm is always centered and marked with either the positive or negative discriminative stimulus odor, and the other arm is always unscented. Digging pots include an accessible and an inaccessible compartment separated by a perforated plastic barrier.Both pots in the apparatus always contain the high-and low-value food, but treats are made accessible depending on the arm and trial type. During positive training trials, the mouse can choose to explore and dig in the scented arm, which contains a pot marked with a positive discriminative stimulus odor and a buried high-value food reward. The mouse can also choose to dig in the unscented arm, which is marked with distilled water and contains a low-value food reward.During negative training trials, The mouse can again choose between the scented arm, which now contains a pot marked with the negative discriminative stimulus odor and no accessible food reward, or the unscented arm, which is again marked with distilled water and contains a low-value food reward. Then, mice are tested in positive and negative un-reinforced trials, where no food rewards are available, and those who have learned the task will undergo an un-reinforced ambiguous trial. In the ambiguous trial, the scented arm contains a pot marked with a mixture of the positive and negative discriminative stimulus odor cues, making the ambiguous odor, while the unscented arm is marked with only distilled water, as always.Mice reveal their judgment biases in this ambiguous trial. For experimental preparation, using a one-milliliter syringe or micropipette, add mint and vanilla extract into labeled centrifuge tubes. Dilute the extracts 1:4 with distilled water and mix by inverting several times.Make these daily and repeatedly invert before use to ensure that the mixtures are fresh and consistent. For the ambiguous odor mixture, after the mint and vanilla essences have been diluted, add equal volumes to a centrifuge tube to make a 1:1 mixture of the diluted essences. Next, during the reinforced trials, for the positive discriminative stimulus odor pot, place one piece of chow in the inaccessible compartment and bury one piece of banana chip in the accessible area of the pot.For the negative discriminative stimulus odor pot, place one piece of chow and one piece of banana chip in the inaccessible compartment. Then, fill the pot with three centimeters of corn cob bedding. No food rewards will be available in the accessible area of this pot.For unscented pots, place one piece of banana chip in the inaccessible compartment and bury one piece of chow in the accessible area of the pot. During all un-reinforced trial tests, place one piece of chow and one piece of banana chip in the inaccessible compartment. Then, fill the pot with three centimeters of corn cob bedding.No food rewards will be available in the accessible area of this pot. After filling the pots, use a syringe or micropipette to draw 100 microliters of the appropriate odor mixture or distilled water and inject it directly on top of the corn cob in a circular motion. Next, to prepare scent dispensers, place one cotton pad piece in the base of the tissue cassette.Then, using a one-milliliter syringe or micropipette, draw 100 microliters of the appropriate odor mixture or distilled water and inject it onto the cotton piece. Cover the tissue cassette with its lid to enclose the scented cotton and create a scent dispenser. Place the digging pots at the end of the arms of the arena and the scent dispensers at the beginning of each arm.Insert the removable door immediately before the cassette slots to block entry to the arena arms and create the start compartment. Perform all training and testing sessions under red light during the dark and active phase of the mouse. Fast the mouse for one hour in their cage before each training or testing session by removing food from the hopper.On day one of digging training, place the food rewards on top of the three-centimeter corn cob bedding instead of burying them underneath. Progressively bury the rewards deeper under the corn cob over the following four days until they're located at the bottom of the three-centimeter bedding by day five. To start training trials, move the mouse from the transport cage to the start compartment of the arena.Remove the start door, immediately lower the plexiglass lid over the arena, and start the five-minute trial timer. Then, record latency to dig as the time at which the first occurrence of digging is observed and latency to eat as the time at which the first occurrence of eating is observed. For discrimination training, conduct one positive reinforced trial followed by one negative reinforced trial on days one to five.On days 6 to 10, pseudo-randomize the order of trials so that each mouse undergoes two positive and two negative trials per day. In the testing phase, conduct one positive or negative un-reinforced test trial for each mouse daily until the learning criteria are met. Each day, perform one positive and one negative reinforced trial in a randomized order before the video-recorded test trial, and another positive and negative reinforced trial after the test trial.Ensure that the positive and negative test trials are conducted in alternating order across days. To perform a video-recorded un-reinforced test trial, move the mouse from the transport cage to the start compartment of the arena. Then, set up a video camera on a tripod so that both pots at the end of the arms are in view and start recording.Ensure to record the cue card with the mouse blind code and the trial type during video scoring. Remove the start door, immediately lower the plexiglass lid over the arena, and start the two-minute trial timer. After testing, return the mouse to its transport cage.Score the positive and negative test trial videos on the day of testing to assess whether the mice have met the learning criteria. Ensure same day video scoring, since animals who meet learning criteria will undergo ambiguous tests the following day. Using event recording software or a stopwatch, let the researcher who is blind to treatment record each mouse's latency to dig and digging duration in each pot during the first minute of positive and negative test trials.Record latency to dig as the time at which the first occurrence of digging is observed, and digging duration as the total time a mouse spends digging. Compare digging duration in the scented pot between positive and negative trials to determine whether mice can discriminate the task. Learning criteria are met if digging duration in the positive discriminative stimulus odor pot is at least double that for the negative discriminative stimulus odor pot during the first minute of testing.For mice that have met the learning criteria, test responses to the ambiguous odor mixture. Perform one positive and one negative reinforced trial in a randomized order. Then, perform one video-recorded test trial using the ambiguous odor mixture.For data analysis, plot the least-squared means of latency in each trial type to confirm that the ambiguous cue presented was interpreted as intermediate. While assessing judgment bias in both the one and two minutes of testing, the trial type by positive discriminative stimulus-odor interaction was significant. This reveals that mice with mint as their positive discriminative stimulus unexpectedly interpreted the ambiguous odor mixtures as positive, while mice with vanilla as their positive discriminative stimulus treated the same ambiguous odor mixtures as intermediate.This finding indicated that only mice with vanilla as their positive discriminative stimulus met the technical requirement for treating the scent mixture as intermediate between the positive and negative discriminative stimulus. Thus, mice with mint as their positive discriminative stimulus were excluded from subsequent judgment bias analyses. Within mice with vanilla as their positive discriminative stimulus, housing influenced the digging latencies, with conventionally housed animals being slower to dig in ambiguous trials than environmentally enriched mice, but not for positive or negative trials.These pessimistic interpretations of ambiguous cues by conventionally housed mice reflect negative judgment biases indicative of negative affect. While performing this protocol, it's important that technical criterion are met. The mice should meet learning criterion showing they're able to discriminate between cues, and interpret the ambiguous cue as intermediate.The development of this new judgment bias task opens the door to test important hypotheses about mouse affective states with both animal welfare and clinical implications.

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